This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The 600 or so proteases encoded in the human genome are involved in a diversity of biological processes. Some function as nonspecific degradative enzymes associated with protein catabolism, exhaustively cleaving many protein substrates at many sites. In contrast, several others function as selective post-translational modifiers, cleaving only a few protein substrates, usually at only one or a few sites. Apoptosis is an important example of a process regulated by limited intracellular proteolysis. This genetically programmed and non-inflammatory form of cell death is a central component of homeostasis and tissue turnover. Since chemotherapeutics typically kill cells by induction of apoptosis, this process is also highly relevant from a therapeutic standpoint. Unfortunately, comprehensive characterization of protease signaling in complex biological samples such as apoptotic cells is often limited by available proteomic methods. We have established a novel method for global sequencing of proteolytic cleavage sites in complex biochemical mixtures that is based on use of an engineered peptide ligase for selective biotinylation of free protein N-termini and positive enrichment of corresponding N-terminal peptides. In collaboration with the UCSF Mass Spectrometry Facility, we are applying this method to the study of proteolysis in apoptosis, using a variety of cancer cell lines and cytotoxic inducers as model systems. Our goal is to globally characterize how proteolysis in apoptosis varies as a function of time, apoptotic inducer, and cell type. The UCSF Mass Spectrometry Facility has been pivotal in providing the mass spectrometry instrumentation, software, and expertise essential for the success of this work.